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Cellular localization and regulation of cytochrome P450-1B1 in different liver cell populations
84
Posters
1 P/CO3/013 1
THE C-TERMINAL PEPTIDE OF ANTITRYPSIN INCREASES LDL BINDING IN HEPG2 CELLS
(AAT)
S Janciauskiene. S .Lindpren
Modulation of Proliferation and Apoptosis in Hepatic Stellate Cells by TGF-P and/ or TNF-a is dependent on the Status of Activation
Gatroenterology-Hepatology Division, Department of Medicine, Malmij University Hospital, Malmb, Sweden
B. Saile. N. Matthes. T. KnitteL G. Ramad ‘. University of GMneen. DeDartment of’&troenterolonv I- and Endocri&logy, G&t&e& G&y
Hydrophobic fragments from various proteolytically degraded precursor proteins may form amyloid fibrils with biological effects unrelated to precursor function. We examined the effects of a hydrophobic 4 kDa C-terminal peptide of AAT (C-36) generated during the cleavage of the AAT molecule by target proteinase on LDL catabolism in HepG2 cells. Results.The soluble form of C-36 increases LDL binding up to 98% while the fibrillar form stimulates LDL binding up to 15 times. This effect was diminished by preincubating the cells with LDL before adding the fibrils or by coincubation of the fibrils with increbing amounts of LDL in cell culture, but was not changed in the presence of serpin-enzyme complexes, suggesting that fibrils affect LDL binding via LDL receptors, and not serpin-enzyme complex (SEC) receptors, or lipoprotein-related protein (LRP) receptors. Analysis of nuclear DNA fragmentation based on the TUNEL assay showed increased amount of fluorescein-labeled DNA in cells incubated with C-36 fibrils relative to controls or cells incubated with C-36 fibrils in the presence of LDL. Moreover. after 4 davs incubation with C-36 fibrils. the cells decreased’ ‘H-thymid;ne incorporation by 60% realtive to controls, but showed no decrease when incubated with tibrils in the presence of LDL. Conclusions. AAT fibril interaction with LDL receptors increases LDL catabolism in hepatocytes which provides a possible explanation for hypocholesterolemia evoked by inflammatory responses. Furthermore, this interaction may disturb intracellular cholesterol homeostasis and induce cell death.
Introduction: During activation process hepatic stellate cells (HSC) increasiugly express both CD95 and CD95L shown to be responsible for self-initiation of cell death It left unknown how apoptosis induction overcomes in case of chronic liver diseases ultimately leading in liver fibrosis. We investigated the e&&s of &:Tiptisud TNF;a on proliferation aud apoptosis of HSC. BrdU-Elisa. Prohferation measurement by Flowcyto’metric apoptosis detection by Annex&V/ propidiumiodide staining. Cell cycle analysis. Western-Blot analysis with antibodies against CD95, CD95L, ~53, WAF-1. Results: TGF-P and T’NF-a did not influence proliferative activity of HSC at day 2 (“resting” cells) but inhibited proliferation of HSC at day 7 (“activated” cells). On the other hand both cytokiues showed apoptosis stimulating effects at day 2, but inhibited spontaneous apoptosis at day 7. Expression of CD95 was not modulated by TNF-a and TGF-P, whereas CD95L-expression was slightly upregulated at day 7. Cell cycle analysis of controls showed a shift of cultures iu GdGl-phase (68f5% at day 2) to the Gz-phase (62rt7% at day 7). At day 7 stimulation with TGF-P and TNF-a led to shift of the HSC cultures to the Gl-phase (TGF-P: 62ti%; TNF-a: 68+7%).WAF-1, being involved in Gl-arrest, was regulated ~53 independent. Compared to the controls TGF-P aud TNF-a downregulated WAF-1 at day 2 and stimulated the expression at day 7. Discussion: TNF-a and TGF-P induce a Glarrest of activated HSC (day 7) which leads to au inhibition of spontaneous apoptosis. This suggests that lack of the cytokiues could lead to induction of spontaneous apoptosis process. 1 P/CO3/016 1
CELLULAR LOCALIZATION AND REGULATION OF CYTOCHROME P450-lB1 IN DIFFERENT LIVER CELL POPULATIONS Piscaalia F, Knittel T. Kobold D, Ramadori G. Dept. of Gastroenterology and Endocrinology, G&tingen, Germany Introduction. We recently found that the cytochrome P450 1B 1 (CyplBl), an important enzyme in the activation of diverse carcinogens, is differently expressed in rat hepatic stellate cells (HSC) during in vitro activatio Drocess. Since the cellular iocalii&ionof-CyplBl in liver tissue’has not been established yet and its biological function remains to be clarified, we investigated the presence and possible induction of CyplBl in various rat liver cell types. Methods. Expression of CyplBl was studied in HSC, in Kupfer cells (KC) and in hepatccytes (HEP) at various time points of primary culture and in hepatic myofibroblasts (MF) by Northern blot analysis. Enzyme inducibilily was assessed by incubation of cells with dimetil-benzanthracene (DMBA). The cellular expression of aromatic hydrocarbon-responsive receptor (AHR), a principle component in the DMBA-mediated induction pathway of CyplBl, was studied by RT-PCR. Results. CyplB 1 was expressed at high levels in HSC and at a minor extent in MF, whereas it was not detectable in KC and in HEP. CyplB 1 cxprcssion was downregulated during HSC activation. DMBA strongly stimulated CyplB 1 expression in HSC, but it did not induce expression in KC and HEP. The AHR was constitutively expressed in all cell types. Conclusions. HSC and MF seem to be the major sources of CyplBl expression in the liver and HSC are the only population responsive to DMBA stimulation. The different induction of CyplBl by DMBA in the \.arious cell populations is not explained by a differential expression of AHR, indicating an AHR independent pathway. The CyplBl expression by HSC and MF might suggest more extended biosynthetic activities, beside to matrix metabolism. On the other hand CyplBl metabolism in HSC.
could
be involved
in the vit.A/lipid
SVRVlVAL OF PATIENTS WlTtl HEPATOCELLULAR CARCINOMA (HCC) IS DETERMINED BY TYPE OF LIVER ESTROGEN RECEPTORS. E,Villa,E.Fqntoni, A.Moles, L.Camellini, P.Buttafoco, A.Grottola, l.Ferretti, F.Manenti. Dept. Internal Medicine/Gastroenterology, Univ. Modena, Italy. Spontaneous survival in HCC varies from few months to years. We therefore prospectively evaluated 42 pts with HCC (r)ot eligible for radical therapy havinsat least 2 lesions ~3 cm in the 2 lobes) for 2 yr. from onset, teevaluate: l.whether liver ER status was associated with different survival and 2. the impact on survival of antihormonal therapy with either tamoxifen (TAM) or megestrol (MEG) according to liver ER status. Elghteen pts [II variant (v) ER, 7 wild-type (wt) ER] did not receive therapy while 24 (12 vER,R wfER) received TAM 80mglUay (wtER) or MEG 16Omg/dq:(vER)](l). Type of ER was analyzed as already described (2). Results were analyzed by Kaplan-Meier method (significant when pc.05). Results: survival in untreated pts was significantly lower in those with *R vs wfE& none of the pts with vER being alive at 2 yr. while 4 (57,1%) tith wfER were (p=O.O269). In pts on antihormonal tllerapy, survival at 2 yr. was not signikantly different between ttje 2 groups, 7 (58,3%) pts with vER and 6 (SO,O%)with wfER being 6till alive (p=O.sSS). Conclusiom 1. the presence of VER is associated with a significantly wors%spontaneous survival, in a$reement with the aggressive growth of tumors with VER (1); 2, endocrine therapy influenced favorably the course of disease in pts with VER, in whom survival greatly improved at 2 yr.; 3. tqmoxifen did not improve significantly the survival of pts with d(ER. (grant 60% MURST and Azien& Policlinico). (1) Cancer Res 1996; 56:3883-85;(2) Cancer Res,l995;55,498-500.
Posters
1 P/CO3/013 1
THE C-TERMINAL PEPTIDE OF ANTITRYPSIN INCREASES LDL BINDING IN HEPG2 CELLS
(AAT)
S Janciauskiene. S .Lindpren
Modulation of Proliferation and Apoptosis in Hepatic Stellate Cells by TGF-P and/ or TNF-a is dependent on the Status of Activation
Gatroenterology-Hepatology Division, Department of Medicine, Malmij University Hospital, Malmb, Sweden
B. Saile. N. Matthes. T. KnitteL G. Ramad ‘. University of GMneen. DeDartment of’&troenterolonv I- and Endocri&logy, G&t&e& G&y
Hydrophobic fragments from various proteolytically degraded precursor proteins may form amyloid fibrils with biological effects unrelated to precursor function. We examined the effects of a hydrophobic 4 kDa C-terminal peptide of AAT (C-36) generated during the cleavage of the AAT molecule by target proteinase on LDL catabolism in HepG2 cells. Results.The soluble form of C-36 increases LDL binding up to 98% while the fibrillar form stimulates LDL binding up to 15 times. This effect was diminished by preincubating the cells with LDL before adding the fibrils or by coincubation of the fibrils with increbing amounts of LDL in cell culture, but was not changed in the presence of serpin-enzyme complexes, suggesting that fibrils affect LDL binding via LDL receptors, and not serpin-enzyme complex (SEC) receptors, or lipoprotein-related protein (LRP) receptors. Analysis of nuclear DNA fragmentation based on the TUNEL assay showed increased amount of fluorescein-labeled DNA in cells incubated with C-36 fibrils relative to controls or cells incubated with C-36 fibrils in the presence of LDL. Moreover. after 4 davs incubation with C-36 fibrils. the cells decreased’ ‘H-thymid;ne incorporation by 60% realtive to controls, but showed no decrease when incubated with tibrils in the presence of LDL. Conclusions. AAT fibril interaction with LDL receptors increases LDL catabolism in hepatocytes which provides a possible explanation for hypocholesterolemia evoked by inflammatory responses. Furthermore, this interaction may disturb intracellular cholesterol homeostasis and induce cell death.
Introduction: During activation process hepatic stellate cells (HSC) increasiugly express both CD95 and CD95L shown to be responsible for self-initiation of cell death It left unknown how apoptosis induction overcomes in case of chronic liver diseases ultimately leading in liver fibrosis. We investigated the e&&s of &:Tiptisud TNF;a on proliferation aud apoptosis of HSC. BrdU-Elisa. Prohferation measurement by Flowcyto’metric apoptosis detection by Annex&V/ propidiumiodide staining. Cell cycle analysis. Western-Blot analysis with antibodies against CD95, CD95L, ~53, WAF-1. Results: TGF-P and T’NF-a did not influence proliferative activity of HSC at day 2 (“resting” cells) but inhibited proliferation of HSC at day 7 (“activated” cells). On the other hand both cytokiues showed apoptosis stimulating effects at day 2, but inhibited spontaneous apoptosis at day 7. Expression of CD95 was not modulated by TNF-a and TGF-P, whereas CD95L-expression was slightly upregulated at day 7. Cell cycle analysis of controls showed a shift of cultures iu GdGl-phase (68f5% at day 2) to the Gz-phase (62rt7% at day 7). At day 7 stimulation with TGF-P and TNF-a led to shift of the HSC cultures to the Gl-phase (TGF-P: 62ti%; TNF-a: 68+7%).WAF-1, being involved in Gl-arrest, was regulated ~53 independent. Compared to the controls TGF-P aud TNF-a downregulated WAF-1 at day 2 and stimulated the expression at day 7. Discussion: TNF-a and TGF-P induce a Glarrest of activated HSC (day 7) which leads to au inhibition of spontaneous apoptosis. This suggests that lack of the cytokiues could lead to induction of spontaneous apoptosis process. 1 P/CO3/016 1
CELLULAR LOCALIZATION AND REGULATION OF CYTOCHROME P450-lB1 IN DIFFERENT LIVER CELL POPULATIONS Piscaalia F, Knittel T. Kobold D, Ramadori G. Dept. of Gastroenterology and Endocrinology, G&tingen, Germany Introduction. We recently found that the cytochrome P450 1B 1 (CyplBl), an important enzyme in the activation of diverse carcinogens, is differently expressed in rat hepatic stellate cells (HSC) during in vitro activatio Drocess. Since the cellular iocalii&ionof-CyplBl in liver tissue’has not been established yet and its biological function remains to be clarified, we investigated the presence and possible induction of CyplBl in various rat liver cell types. Methods. Expression of CyplBl was studied in HSC, in Kupfer cells (KC) and in hepatccytes (HEP) at various time points of primary culture and in hepatic myofibroblasts (MF) by Northern blot analysis. Enzyme inducibilily was assessed by incubation of cells with dimetil-benzanthracene (DMBA). The cellular expression of aromatic hydrocarbon-responsive receptor (AHR), a principle component in the DMBA-mediated induction pathway of CyplBl, was studied by RT-PCR. Results. CyplB 1 was expressed at high levels in HSC and at a minor extent in MF, whereas it was not detectable in KC and in HEP. CyplB 1 cxprcssion was downregulated during HSC activation. DMBA strongly stimulated CyplB 1 expression in HSC, but it did not induce expression in KC and HEP. The AHR was constitutively expressed in all cell types. Conclusions. HSC and MF seem to be the major sources of CyplBl expression in the liver and HSC are the only population responsive to DMBA stimulation. The different induction of CyplBl by DMBA in the \.arious cell populations is not explained by a differential expression of AHR, indicating an AHR independent pathway. The CyplBl expression by HSC and MF might suggest more extended biosynthetic activities, beside to matrix metabolism. On the other hand CyplBl metabolism in HSC.
could
be involved
in the vit.A/lipid
SVRVlVAL OF PATIENTS WlTtl HEPATOCELLULAR CARCINOMA (HCC) IS DETERMINED BY TYPE OF LIVER ESTROGEN RECEPTORS. E,Villa,E.Fqntoni, A.Moles, L.Camellini, P.Buttafoco, A.Grottola, l.Ferretti, F.Manenti. Dept. Internal Medicine/Gastroenterology, Univ. Modena, Italy. Spontaneous survival in HCC varies from few months to years. We therefore prospectively evaluated 42 pts with HCC (r)ot eligible for radical therapy havinsat least 2 lesions ~3 cm in the 2 lobes) for 2 yr. from onset, teevaluate: l.whether liver ER status was associated with different survival and 2. the impact on survival of antihormonal therapy with either tamoxifen (TAM) or megestrol (MEG) according to liver ER status. Elghteen pts [II variant (v) ER, 7 wild-type (wt) ER] did not receive therapy while 24 (12 vER,R wfER) received TAM 80mglUay (wtER) or MEG 16Omg/dq:(vER)](l). Type of ER was analyzed as already described (2). Results were analyzed by Kaplan-Meier method (significant when pc.05). Results: survival in untreated pts was significantly lower in those with *R vs wfE& none of the pts with vER being alive at 2 yr. while 4 (57,1%) tith wfER were (p=O.O269). In pts on antihormonal tllerapy, survival at 2 yr. was not signikantly different between ttje 2 groups, 7 (58,3%) pts with vER and 6 (SO,O%)with wfER being 6till alive (p=O.sSS). Conclusiom 1. the presence of VER is associated with a significantly wors%spontaneous survival, in a$reement with the aggressive growth of tumors with VER (1); 2, endocrine therapy influenced favorably the course of disease in pts with VER, in whom survival greatly improved at 2 yr.; 3. tqmoxifen did not improve significantly the survival of pts with d(ER. (grant 60% MURST and Azien& Policlinico). (1) Cancer Res 1996; 56:3883-85;(2) Cancer Res,l995;55,498-500.